Steps involved in in vitro regeneration of a complete plants. Regeneration refers to the development of organised structures like roots, shoots, flower buds, somatic embryos (SEs), etc. from cultured cells/tissues; the term organogenesis is also used to describe these events. Root regeneration occurs quite frequently but it is useful only in case of shoots and embryo germination. Only shoot and SE regenerations give rise to complete plants which is essential for applications of tissue culture technology in agriculture and horticulture. Regeneration may occur either directly from the explant or may follow an intervening callus phase.
Basic technique of Plant Tissue Culture: The basic technique of plant tissue culture involves the following steps:
• Preparation and sterilisation of suitable nutrient medium : Suitable nutrient medium as per objective of culture is prepared and transferred into suitable containers. Culture- medium is rich in sucrose, minerals, vitamins, and hormones. Yeast extract, coconut milk are also added. The culture is completely sterilized in an autoclave.
• Selection of explants : Selection of explants such as shoot tip should be done.
• Sterilisation of explants: Surface sterilisation of the explants by disinfectants (e.g., sodium hypochlorite or mercuric chloride) and then washing the explants with sterile distilled water is essential.
• Inoculation: Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilised by autoclaving to avoid microbial contamination) in culture vessels under sterile conditions is done.
• Incubations : Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light: 16 hours of photoperiod), temperature (- 26°C) and relative humidity (50 – 60%) is required.
• Regeneration : An unorganized mass of cells developing from explants is called callus. The callus gives rise to embryoids which can develop into whole plant if the medium is provided with proper concentration of hormones. This property’ of developing every somatic cell into a Ml fledged plant is called totipotenev. Regeneration of plant, from cultured plant tissues is carried out.
• Hardening: Hardening is gradual exposure of plantlets to an environmental conditions.
• Plantlet transfer : After hardening, plantlets are transferred to the greenhouse or field conditions following acclimatization (hardening) of regenerated plants.
